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| Status |
Public on Mar 07, 2012 |
| Title |
H2BUb1_ChIP-seq |
| Sample type |
SRA |
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| Source name |
NCCIT, H2BUb1 ChIP
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| Organism |
Homo sapiens |
| Characteristics |
cell line: NCCIT
cell type: embryonic carcinoma cells
chip antibody: anti-H2BUb
antibody vendor: Millipore
antibody catalog number: 17-650
antibody lot#: NG1606340
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| Growth protocol |
NCCIT embryonic carcinoma cell lines on a 10cm dish were cultured in RPMI 1640 supplemented with 10% fetal bovine serum (FBS, Hyclone) and 1% antibiotic-antimycotic solution (Hyclone) at 37ºC in a humidified atmosphere composed of 5% CO2.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Genomic DNA extraction: Cross-linking was performed by incubating cells in 1% formaldehyde (Sigma) for 20 min at 25°C. Cells were quenched with 125 mM Glycine. PBS-washed cells were lysed in 400 μl of SDS Lysis Buffer, and DNA was sonicated to create fragments of 400-500 nucleotides in length using a Sonic Dismembrator (Model 500, Fisher).
Chromatin immunoprecipitation: Chromatin cleared by centrifugation for 15 minutes at 13,000 rpm, 4°C were subjected to immunoprecipitation using antibodies of interest with a mixture of protein A and G agarose (GE Healthcare) for 2 hr at 4°C. Protein A and G agarose/antibody/chromatin complexes then were washed for 10 minutes with each of the following solutions: Low Salt Wash Buffer (150 mM NaCl lysis buffer without 0.2% SDS), High Salt Wash Buffer (500 mM NaCl lysis buffer without 0.2% SDS), LiCl buffer (10 mM Tris-Cl pH 8.0, 250 mM LiCl, 1 mM EDTA, 0.5% NP-40, 0.5% Sodium deoxycholate), and TE buffer (10 mM Tris-Cl pH 7.5, 1 mM EDTA). The chromatin complexes were then eluted twice in 250 μl elution buffer (1% SDS, 0.1M NaHCO3) by incubation at 25°C for 15min with rotation and were reverse-crosslinked by heating at 68ºC for O/N with 400mM NaCl.
Antibodies: Antibodies recognizing H3ac, H3K4me3, H3K36me3, H3K79me1, H3K79me2, H3K79me3 were polyclonal rabbit antibodies purchased from Abcam or produced in-house. H2Bub antibody was obtained from Millipore (17-650).
The purified ChIP DNA fragments were ligated to a pair of adaptors for sequencing on the Illumina Genome Analyzer. The ligation products were size-fractioned to obtain 200- to 300-bp fragments on an agarose gel and subjected to 18 cycles of PCR amplification. Cluster generation and 72 cycles of single-read sequencing were done.
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina Genome Analyzer |
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| Data processing |
The sequence tags were mapped to the human genome (UCSC hg18) by using the Genome Analyzer data analysis pipeline. The mapped sequence reads were extended to the average size of fragment length (200bp).
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| Submission date |
Dec 21, 2010 |
| Last update date |
May 15, 2019 |
| Contact name |
Inkyung Jung |
| E-mail(s) |
ijungkaist@gmail.com
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| Organization name |
KAIST
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| Department |
Biological Sciences
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| Street address |
KAIST, 291 Daehak-ro, Yuseong-gu
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| City |
Daejeon |
| ZIP/Postal code |
34141 |
| Country |
South Korea |
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| Platform ID |
GPL9052 |
| Series (1) |
| GSE25882 |
Genome-wide maps of chromatin state in NCCIT cells |
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| Relations |
| SRA |
SRX037080 |
| BioSample |
SAMN00189371 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM644989_Ub_2.bed.gz |
46.8 Mb |
(ftp)(http) |
BED |
SRA Run Selector |
| Processed data provided as supplementary file |
| Raw data are available in SRA |
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